D-methionine (Met) relative to L-Met for nursery pigs using the slope-ratio assay. A total of 50 crossbred barrows with an initial BW of 13.5 kg (SD = 1.0) were used in an N balance study. A Met-deficient basal diet (BD) was formulated to contain an adequate amount of all amino acids (AA) for 10–20 kg pigs except for Met. The two reference diets were prepared by supplementing the BD with 0.4 or 0.8 g L-Met/kg at the expense of corn starch, and an equivalent concentration of D-Met was added to the BD for the two test diets. The pigs were adapted to the experimental diets for 5 d and then total but separated collection of feces and urine was conducted for 4 d according to the marker-to-marker procedure. Nitrogen intakes were similar across the treatments. Fecal N output was not affected by Met supplementation regardless of source and consequently apparent N digestibility did not change. Conversely, there was a negative linear response (P < 0.01) to Met supplementation with both Met isomers in urinary N output, which resulted in increased retained N (g/4 d) and N retention (% of intake). No quadratic response was observed in any of the N balance criteria. The estimated bioavailability of D-Met relative to L-Met from urinary N output (g/4 d) and N retention (% of intake) as dependent variables using supplemental Met intake (g/4 d) as an independent variable were 87.6% and 89.6%, respectively; however, approximately 95% of the fiducial limits for the relative bioavailability estimates included 100%. In conclusion, with an absence of statistical significance, the present study indicated that the mean relative bioequivalence of D- to L-Met was 87.6% based on urinary N output or 89.6% based on N retention.This experiment was conducted to determine the bioavailability of  D- and L-Met which is produced through chemical synthesis  are commonly used to provide indispensable AA , which limit growth of pigs when protein sources are marginally used to reduce feed cost as well as N excretion. Methionine is essential for protein synthesis and is one of the most limiting AA for the growth of nursery pigs fed diets containing dried blood products (plasma and cells) and dried whey . Methionynthesis , but onl in pigs . Thus, iurements . The claurements  and is mreported  to the aThe Institutional Animal Care and Use Committee of Konkuk University reviewed and approved all protocols (KU13188) used in the present study.D- to L-Met during five consecutive periods. In each 9-d period, 10 pigs were allotted to five dietary treatments with two replicates per treatment in a randomized complete block design based on the initial BW.A total of 50 crossbred ((Landrace × Yorkshire × Duroc) × Duroc) nursery barrows at 35 ± 3 d of age were selected based on BW which was similar across the pigs. For adaptation to metabolism cages, the pigs were individually placed in the cages for five days. After adaptation, the pigs (13.5 ± 1.0 kg) were used to estimate the relative bioavailability of L-Met/kg at the expense of corn starch, and an equivalent concentration of D-Met was added to the BD for two test diets. To avoid any possible variation in the experimental diets caused by feed ingredients, the experimental diets were made of the same batch of ingredients, mixed at the same time and at the same location. To minimize orts, daily feed allowance was calculated as 3.5% of the BW of each animal at the beginning of each period. The feed was divided into two equal meals and fed to the pigs at 900 and 1,700 h. The pigs were provided ad libitum access to water.A Met-deficient basal diet (BD) was formulated to meet or exceed the estimated requirements of all nutrients except for Met. The BD contained total Met at 18.1 g/kg which was about 66% of the Met requirement for 10–20 kg pigs ; Table 1For the N-balance study, the pigs were adapted to the experimental diets for 5 d and then total but separated collection of feces and urine was conducted for 4 d according to the marker-to-marker procedure . The colAt the completion of the study, the frozen fecal samples were dried in a forced-air oven at 55 °C and finely ground prior to chemical analyses. The experimental diets, fecal and urine samples were determined for crude protein (CP) content (N × 6.25) by the Kjeldahl method .I is the amount of N ingested (g); NF and NU are the amount of N voided via the feces (g) and urine (g), respectively.Apparent total tract N digestibility and retention were calculated using the following equations:D-Met to L-Met was estimated using a multiple regression model and the slope-ratio analysis described by a is intercept; e is random error; sb and tb are the slopes for L- and D-Met, respectively; sx and tx are the concentrations of L- and D-Met intake, respectively. An individual pig served as the experimental unit and statistical significance was determined at P < 0.05.Experimental data were analyzed using the MIXED procedures of SAS . The independent variables in the model included diet as a fixed effect and period and block nested within period as random effects. The orthogonal polynomial contrast was used to examine the relationship between N balance response criteria and graded concentrations of Met isomers. The relative bioavailability of L- or D-Met on N balance are shown in P < 0.05) to Met supplementation from L- or D-Met in urinary N output, which resulted in increased (P < 0.01) retained N and N retention. No quadratic response was observed in any of the N-balance criteria. The estimated bioavailability of D-Met relative to L-Met from urinary N output (g/4 d) and N retention (% of intake) as dependent variables using supplemental Met intake (g/4 d) as an independent variable were 87.6 and 89.6%, respectively  and N retention (% intake) as dependent variables, are 87.6% and 89.6%, respectively, and the 95% fiducial limits included 100%. To obtain accurate values from bioavailability assays, the assumptions for slope-ratio assay should be validated  was used as the response criterion. This was in agreement with L- or DL-Met and reported no difference in utilization between Met isomers. Furthermore, no differences in BW gain or plasma urea levels were observed when three-week-old pigs received low-protein-liquid diets containing either DL- or L-Met (0.51%) for seven days  and N retention (% of intake) were 87.6% and 89.6%, respectively; but 95% fiducial limits for the relative bioavailability estimates included 100%.In conclusion, the relative bioavailability values for 10.7717/peerj.2368/supp-1Supplemental Information 1Click here for additional data file.


Salmonella infections in newly hatched chicks result in enteric and systemic diseases with a high mortality. Probiotics can improve the health of a host. The purpose of the present study was to investigate the effect of Lactobacillus plantarum LTC-113 on the gut permeability in the presence or absence of Salmonella  infection. Newly hatched chicks were randomly allocated to 4 treatments (i) NC (negative control); (ii) LAC (the L. plantarum LTC-113-treated group); (iii) SAL , and (iv) LAC + SAL . Compared with the NC group, the intestinal permeability and claudin-2 (CLDN-2) were significantly increased, while mRNA levels of zonula occludens-1 (ZO-1) and claudin-5 (CLDN-5) were significantly decreased in the SAL group. However, these changes were eliminated in the LAC + SAL group. Additionally, numbers of Salmonella in liver, spleen and ceca were significantly reduced in the LAC + SAL group compared with the SAL group. Moreover, L. plantarum LTC-113 prevented the increase of inflammatory meditators myeloperoxidase (MPO), LITAF, IL-1β, IL-6 and inflammation scores induced by Salmonella. These findings indicate that L. plantarum LTC-113 can protect hosts from Salmonella induced intestinal barrier disruption by regulating expression of tight junction genes and inflammatory meditators and decreasing Salmonella colonization. Salmonella is one of the most common gastrointestinal pathogens in chickens. To date, more than 2000 serovars of Salmonella have been identified. Chickens can be colonized by numerous serovars of Salmonella. Among them, S. Pullorum and S. Gallinarum are specific for chickens. On the other hand, other serovars, such as S. Typhimurium, S. Enteritidis and S. Heidelberg, have a broad range of hosts and are most commonly associated with human infections1. While S. Typhimurium is not the most prevalent in poultry clinical cases, it is certainly one of the most important isolates. For example, Lamas et al.2 investigated the prevalence of Salmonella isolated from broiler houses between 2011 and 2015 and reported that S. Typhimurium was the most prevalent Salmonella among sixteen different serotypes found.The genus Salmonella cause over 90 million cases of Salmonellosis worldwide every year; about 85% of which are caused by Salmonella contaminated food3. In the United States, the total annual cost of foodborne salmonellosis was about $3.6 billion in 20134. Poultry are considered one of the most important Salmonella reservoirs. Exposure to contaminated poultry-derived foods, mainly eggs and egg products, but also chicken meat, is one of the main reasons for Salmonellosis in humans. Therefore, preventing the Salmonella infection in poultry may be an effective measure to decrease salmonellosis in humans.Salmonella infections occur via oral transmission where this pathogen must overcome several intestine barriers in the host to finally interact with the intestinal epithelium and thereby penetrate into deeper tissues. The intestine barriers include the luminal microbiota, a mucus layer, epithelial integrity and immune responses5. Oral infection of newly hatched chicks with S. Typhimurium was capable of inducing the expression of chemokines IL-8 and K60 along with the proinflammatory cytokine IL-1β in intestinal tissues and in the liver, indicative of an early inflammatory response6. However, such responses were not observed in 1 week old birds7. The different responses between newly hatched chicks and older birds could be mainly due to the following two reasons. Firstly, the gut microbiota in newly hatched chicks is going through a transition from a transient community to one of increasing complexity as the birds age. Proteobacteria are the predominant phylum in one-day old chicks, whereas Firmicutes are the predominant one in older birds (14–28 days of age). The latter also has a significantly more diverse microbial community structure8. Secondly, the gut-associated lymphoid tissue contains functionally immature T and B lymphocytes at the time of hatching, indicative of immature immunity of the newly hatched chicks9. Different cellular junctions, including tight junctions (TJ), adherence junctions (AJ), desmosomes and gap junctions, work together to maintain the integrity of the epithelial barrier10. Apical junctional complexes (AJC), consisting of TJ and AJ, are the most important in maintaining permeability of the epithelium by selectively restricting paracellular diffusion11. Previous studies have shown that broilers challenged with S. Typhimurium at 7 days of age exhibited decreased expression of CLDN-1, occludin, and mucin-2 transcripts at day2112. The in vivo results supported previous in vitro findings that S. Typhimurium decreased expression of CLDN-1, ZO-1, and E-cadherin and influenced the distribution of these proteins in Caco-2 cells15.16, through regulation of cellular junctions. Hummel et al.11 found that four Lactobacilli, including L. acidophilus, L. fermentum, L. gasseri, and L. rhamnosus, strongly increased the transepithelial resistance and modulated the gene expression of adherence junction proteins such as E-cadherin and β-catenin in T84 cells. Mennigen et al.17 found that the probiotic mixture VSL#3 ameliorated the leakiness of the colonic epithelium to Evans blue and prevented the decrease in expression and redistribution of the tight junction proteins occludin, ZO-1, and CLDN-1, -3, -4, and -5 in dextran sodium sulfate (DSS)-induced colitis mice. Another way by which probiotics enhance epithelial barrier functions is to inhibit the cell apoptosis. The probiotics mixture VSL#3 decreased apoptosis in a murine model of colitis17. Nevertheless, the effects of probiotics on the intestinal barrier function of newly hatched chickens challenged with Salmonella have not been reported. Therefore, we hypothesized that L. plantarum LTC-113 could restore the intestinal barrier disrupted by Salmonella infection in newly-hatched chicks. The purpose of the present study was to investigate the effect of L. plantarum LTC-113 on the gut permeability in the presence or absence of Salmonella infection.One important beneficial effect of probiotics is to enhance epithelial barrier functionsSalmonella challenge, the amounts of S. Typhimurium in ceca, spleen and liver were determined. S. Typhimurium was not detectable in the tissue samples from the NC and LAC groups. In comparison with the SAL group, the number of S. Typhimurium colonized in the liver, spleen and ceca were significantly lower in the LAC + SAL group (P < 0.05)  Fig. .Figure 1S. Typhimurium into spleen and liver was associated with altered intestinal permeability, intestinal paracellular permeability was determined by measuring DX-4000-FITC in plasma. As showed in Fig. S. Typhimurium infection significantly increased the intestinal permeability as compared with the NC group (P < 0.05). However, the intestinal permeability of chicks in the LAC + SAL group was significantly lower than that in the SAL group (P < 0.05). There was no significant difference between the LAC group and the LAC + SAL group (P > 0.05).To determine whether translocation of S. Typhimurium and L. plantarum LTC-113, expression of selected AJ and TJ genes was measured by RT-PCR. As showed in Fig. P > 0.05). In comparison with the NC group, Salmonella infection (the SAL group) significantly decreased mRNA levels of ZO-1 and CLDN-5 and increased the mRNA level of CLDN-2 (P < 0.05). The down-regulation of ZO-1 and CLDN-5 and the up-regulation of CLDN-2 due to Salmonella infection were eliminated in the LAC + SAL group. Moreover, the mRNA expression level of ZO-1 in the LAC group was significantly higher than that in the NC group (P < 0.05) . L. plantarum LTC-113 pretreatment (LAC + SAL) significantly decreased the inflammation score and total score compared with the SAL group (P < 0.05), but the crypt damage score was not affected by L. plantarum LTC-113 (P > 0.05)   Fig. . Apoptot05) Fig. . However05) Fig. .Figure 4P < 0.05). In comparison with the SAL group, L. plantarum LTC-113 pretreatment (LAC + SAL) significantly decreased the activities of MPO (P < 0.05)  Fig. .Figure 6Salmonella infection, levels of cytokines LITAF, IL-1β, and IL-6 in serum samples were determined by ELISA  significantly increased the levels of LITAF, IL-1β, and IL-6 in serum compared with the NC group (P < 0.05). However, the levels of LITAF, IL-1β, and IL-6 in the LAC + SAL group were significantly lower than in the SAL group (P < 0.05).To further study inflammatory responses induced by ISA Fig. . SalmoneSalmonella easily colonize and act on the intestinal epithelium of newly hatch chicks. The gut of a chick during the first 3 days is only protected by high expression of β-defensins18. An activation and normalization of the innate immune system, characterized by high expression of cytokines IL-8 and IL-17, starts on day 4 post-hatch18.Chicks at one day old were used in this study, since they have a low diversity and density of gut microbiota and an immature immunity. S. Typhimurium infection on the epithelial integrity of newly hatched chicks by measuring permeability of DX-4000-FITC. In this study, Salmonella infection was accompanied by a loss of epithelial integrity, as indicated by increased permeability of DX-4000-FITC. Zhang et al.12 reported that S. Typhimurium infection caused intestinal injury in broiler chickens, as determined by an increase in plasma endotoxin levels. Moreover, disruption of the intestinal epithelial barrier by S. Typhimurium was also reflected by translocation of S. Typhimurium into spleen and liver in our current study. Our result was in agreement with the studies of Zhang et al.19 and Feng et al.20 in chicks. In addition, Köhler et al.13 found that infection of human intestinal T84 cells by S. Typhimurium over 2 h resulted in an 80% loss of transepithelial electrical resistance, increased bacterial translocation and initiation of polymorphonuclear leukocyte migration across the intestinal barrier and increased the paracellular influx of inulin. Similarly, Yu et al.15 found that S. Typhimurium SL1344 increased the dextran permeability and decreased the transepithelial electrical resistance in Caco-2 cells. Streptomycin-pretreated mice challenged with Salmonella resulted in translocation of Salmonella into the liver21. Li et al.22 also found that Salmonella damaged intestinal structure severely of mice and resulted in the increase of bacteria translocation into the mesenteric lymph node. These results demonstrate that Salmonella infections can disrupt the intestinal permeability of hosts.To the best of our knowledge, this study is the first to evaluate the effect of 11. Both junction complexes are formed by various transmembrane proteins. The core of TJ complexes is composed of occludin, ZO and CLDN proteins, while the core of AJ complexes is composed of β-catenin and E-cadherin. Therefore, to better clarify the molecular mechanism of S. Typhimurium induced loss of intestinal epithelial barrier functions, changes in the gene expression of 5 transmembrane proteins  and 3 intracellular proteins  of AJ and TJ at the mRNA level were determined.The paracellular pathway of intestinal cell is mainly modulated by cell junction complexes TJ and AJS. Typhimurium infection up-regulated the gene expression of CLDN-2 and down-regulated the gene expression of ZO-1 and CLDN-5. Claudins are mainly responsible for linking adjacent enterocytes through interactions of their extracellular loops in TJ complexes. CLDN-5 is known to form tight junctions and decrease permeability, whereas CLDN-2 stimulates expression of cation-selective pores and increases the permeability23. The ZO-1 scaffolding proteins play an important role in linking transmembrane junctional proteins to the actomyosin cytoskeleton and several cytoplasmic regulatory proteins24. In this study, an increase in CLDN-2 and reductions in the expression of ZO-1 and CLDN-5 induced by S. Typhimurium disrupted the intestinal barrier, consequently enabling S. Typhimurium translocation into the spleen and liver. Köhler et al.13 reported that T84 monolayers infection with S. Typhimurium dramatically decreased expression of ZO-1 and led to loss of the barrier function. Yu et al.15 and Shao et al.25 also reported that Salmonella infection decreased expression of ZO-1 protein and caused a large increase in permeability in Caco-2 cells. Zhang et al.19 found that Salmonella infection increased the leaky protein CLDN-2, permeability and bacterial translocation in the human intestinal epithelial cell lines SKCO15 and HT29C19A as well as in streptomycin-pretreated C57BL6 mice. Moreover, studies have shown that TNF-α, and ILs also could regulate the intestinal barrier26. Cui et al.27 found that TNF-α could decrease the level of phosphorylation of CLDN-1, and dissociate CLDN-1 from TJs. TNF-α has not been found nor described in the broiler genome. However, LITAF is responsible for the expression of TNF-α in mammals28 and has been reported to play an important role in intestinal inflammation of broilers29. Suzuki et al.30 reported that IL-6 could cause the upregulation of CLDN-2, thus increasing mucosal permeability. In this study, Salmonella elevated the levels of LITAF and IL-6, this may be associated with the increase of permeability induced by Salmonella. Taken together, these results indicated that S. Typhimurium induced the increase of permeability by disrupting the structure of TJ and AJ complexes.Our results showed that 31. In our study Salmonella infection did not cause an increase in intestinal cell apoptosis. This result disagrees with previous reports, possibly due to the fact that only the first 8 hours of Salmonella infection was studied. Santos et al.32 found that healthy male rhesus macaques challenged with Salmonella resulted in an increase in intestinal mucosa cell death. In an in vitro study, Li et al.33 reported that apoptosis was induced by Salmonella 12 hours after infection in Caco-2 cells. Paesold et al.34 found that apoptosis of epithelial cells infected with Salmonella was delayed for approximately 28 h after bacterial entry. Taken together, all the results suggest that Salmonella infection disrupts epithelial integrity mainly by interfering with the TJ and AJ complexes at the early stage and by apoptosis at the later stage.Besides changes in tight junction complexes, enhanced cell apoptosis is also associated with disruption of epithelial integrityin vitro on epithelial monolayers and in vivo rat models have demonstrated that probiotics could improve barrier function17. In this study, administration of L. plantarum LTC-113 abolished the change of ZO-1, CLDN-5, and CLDN-2 induced by Salmonella. Meanwhile, L. plantarum LTC-113 eliminated the increased permeability and bacterial translocation induced by Salmonella. Qin et al.35 found that Lactobacillus plantarum could alleviate Salmonella-induced increase of dextran permeability and decrease of ZO-1 proteins in Caco-2 cells. Yu et al.15 found that Lactobacillus. amylophilus D14 protected Caco-2 cells from S. Typhimurium SL1344 induced increase of dextran permeability and decrease of ZO-1 proteins in Caco-2 cells. All above results indicate that L. plantarum LTC-113 protected the intestinal epithelial barrier from Salmonella infection through regulating expression of TJ and AJ proteins. In addition, the number of Salmonella in the intestinal was reduced by administration of L. plantarum LTC-113 in Salmonella infection chicks, which could be another mechanism by which L. plantarum LTC-113 protected the intestinal barrier integrity.Probiotics have been defined as living bacteria that, when ingested in sufficient quantity, improve the health of the host. Studies Salmonella infection induced the damage of intestinal morphology in this study. The inflammation scores were elevated by Salmonella infection and L. plantarum LTC-113 could alleviate the damage of intestinal morphology and decrease inflammation scores. This result was accompanied by the change of myeloperoxidase (MPO) and proinflammatory cytokines LITAF, IL-1β, and IL-6. MPO is a leukocyte-derived enzyme and is an important indicator of inflammatory responses. In the present study, the increase in serum MPO activities of the SAL group suggested that Salmonella infection could activate monocytes and neutrophils in blood and promote intestinal inflammations. However, the decrease of MPO activities in the LAC + SAL group demonstrated that L. plantarum LTC-113 could depress Salmonella induced inflammatory responses. This was also supported by the results of cytokines LITAF, IL-1β, and IL-6. LITAF of chickens29. IL-1β is indicative of inflammation and is mainly secreted by monocytes, tissue macrophages and other cells36. IL-6 is associated with secretion of acute phase proteins that are involved in inflammatory responses37. In this study, L. plantarum LTC-113 prevented the increase of proinflammatory cytokines induced by Salmonella infection. Feng et al.20 have also demonstrated similar changes. Our results demonstrate that Salmonella infection could induce intestinal inflammation of newly hatched chicks by increasing the levels of inflammatory meditator myeloperoxidase and proinflammatory cytokines LITAF, IL-1β, and IL-6, and L. plantarum LTC-113 could alleviate intestinal inflammation induced by Salmonella in newly hatched chicks.The Salmonella by the gut microbiota and the innate immune system are important for a host to prevent Salmonella from interacting with intestinal epithelium38. The gut microbial composition of young children continues to develop until 3 years of age39. Moreover, infants are more susceptible to Salmonella infections, due to the immaturity of their immune systems. These two phenomena exist in other young animals, especially in newly hatched chickens. In commercial poultry production, hatching is carried out in a clean incubator. Newly hatched chicks, unlike other mammals, will not have an access to maternal antibodies and healthy microbiota via contact with adult chickens. Consequently, the gut microbiota in newly hatched chickens is under-developed as indicated by low diversities and densities42. Moreover, the immunity of the newly hatched chicken is immature9. In this study, we demonstrate that S. Typhimurium infections could disrupt intestinal permeability and induce cecal inflammation. Thus, newly hatched chicks may be used as another model for studying Salmonella infections in young children.Resistance to colonization of Salmonella disrupted the intestinal epithelial barrier in newly hatched chicks by increasing expression of CLDN-2 and decreasing expression of ZO-1 and CLDN-5, thereby allowing bacterial translocation. Moreover, L. plantarum LTC-113 could protect newly hatched chicks from Salmonella induced intestinal epithelial barrier disruption by stabilizing the expression of tight junction genes, regulating the levels of inflammatory meditators, and decreasing the Salmonella colonization.In summary, L. plantarum LTC-113 treatment and no Salmonella infection, NC); (ii) the L. plantarum LTC-113-treated group ; (iii) the Salmonella-infected group ; and (iv) the L. plantarum LTC-113-treated and Salmonella-infected group . All chicks had free access to water and a starter feed without antibiotics during the experiment. All experimental protocols used in this experiment were in accordance with those approved by the Northwest Agriculture and Forestry (A&F) University Institutional Animal Care and Use Committee (protocol number NWAFAC1036) and the institutional safety procedures were followed.Eighty 1-day-old healthy male Nick chicks were purchased from a local hatchery . The chicks were randomly divided into four groups. The treatment groups were as follows: (i) the negative control (no L. plantarum) was selected based on preliminary in vitro and in vivo results for this study. To prepare the LAB inoculum, L. plantarum strain LTC-113 was propagated twice in the DeMan, Rogosa, and Sharpe (MRS) broth at 37 °C without shaking. The number of colony-forming units (CFU) in culture was measured by plating on MRS plates after a series dilution.The potential probiotic strain used in this study was isolated by our lab. Briefly, lactic acid bacteria (LAB) were isolated from intestinal contents of Tibet chickens and confirmed by sequencing of 16 s rRNA and comparison with the GenBank database. Among 139 isolated lactic acid bacteria strains, LTC-113 strain . S. Typhimurium CVCC542 was grown overnight in the Luria-Bertani (LB) broth at 37 °C in an orbital shaking incubator at 180 rpm/min, sub-cultured twice, and then the CFU was measured by plating on LB plates after a series dilution.A spontaneous novobiocin-resistant of 9 CFU of the L. plantarum LTC-113, 1 × 109 CFU of the S. Typhimurium CVCC542, 1 × 109 CFU of the L. plantarum LTC-113 and 1 × 109 CFU of the S. Typhimurium CVCC542 via oral gavage, respectively. The chicks in the NC group received 0.2 ml of sterile PBS.On the first day of age, chicks in groups LAC, SAL and LAC + SAL received 0.2 ml phosphate-buffered saline (PBS) containing 1 × 10Salmonella infection, 6 chicks from each group were randomly selected. Blood samples were collected from the carotid vein. For the separation of blood sera, blood samples were incubated for 1 h at room temperature followed by centrifuging at 2,000 g for 10 min. After blood collection, chickens were euthanized by cervical dislocation. One arm of cecum, liver and spleen were collected for bacterial enumeration. Meantime, a segment (2 cm) from one arm of the cecum  was collected and separated into two parts. One part was fixed in 4% paraformaldehyde, while the other part was kept in liquid nitrogen for RNA extraction.Eight hours after the The collected tissue samples  were homogenized in sterile PBS and plated out onto the selective Brilliant Green agar containing 50 μg of novobiocin per ml after a series dilution. Plates were incubated at 37 °C for 24 h before enumeration of the colonies. All microbiological analyses were performed in duplicate and the average numbers of CFU were subjected to logarithmic transformation before statistical analysis. The results were calculated and expressed as log10 colony-forming units per gram tissue.43. Briefly, 5 chicks that had fasted for 4 h were randomly selected from each group and were given DX-4000–FITC  by oral gavage (0.5 mg/g body weight) 8 hours post-infection (hpi). Our preliminary experiment indicated that S. Typhimurium infections induced the highest intestinal permeability of newly hatched chickens at 8 hpi. After 1 h, blood was collected from the carotid vein and was centrifuged at 4 °C, 2, 000 g for 10 min. Fluorescence intensity of 100 μL plasma was measured with a fluorescence spectrophotometer  at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The concentrations of DX-4000-FITC per mL of sera were calculated from a standard curve with known FITC concentrations.The intestinal permeability was measured by using 4000 Da fluorescent dextran–FITC (DX-4000–FITC)  as previously described45 or newly designed with the Primer premier 5.0 software (Table http://medgen.ugent.be/~jvdesomp/genorm/). GAPDH was finally chosen to normalize gene expression due to its high expression stability. The efficiency of all tested genes was between 90% and 110%. Target gene expression was normalized with GAPDH gene expression. The method of 2−ΔΔCt was used to analyze the real-time PCR data46 and results were expressed as the fold change relative to the average value of the negative control group.Total RNA from ceca was extracted by using the Takara Total RNA Kit I  according to the manufacturer’s instructions and quantified by spectrophotometry . First-strand cDNA synthesis was performed using a reverse transcription kit  and 1 μg of extracted total RNA according to the manufacturer’s instructions. The obtained cDNA was stored at −80 °C until use. Quantifications of target genes occludin, CLDN-1, CLDN-2, CLDN-5, ZO-1, ZO-2, E-cadherin, β-catenin and a housekeeping gene ) in cDNA samples were carried out by fluorometric real-time PCR using a Bio-Rad CFX-96 instrument  and real-time PCR kits . Primers for specific genes were either described previouslyet al.17, including the degree of inflammation, the transmural vertical extent of inflammation and the crypt damage score, in relation to the percentage of involvement of mucosal surface in each slide . Histological inflammation was graded by two blinded investigators using a score system introduced by Mennigen in situ detection of cell apoptosis. Five-micrometer sections of the cecum were de-paraffinized, hydrated, and treated with a solution containing proteinase K (20 mg/ml) and 0.5% Triton X-100 . In situ detection of cell apoptosis was performed using a commercial kit  according to the manufacturer’s instructions. Sample slides as positive controls were treated with RNase-free DNase I at room temperature for 10 min before incubation with the TUNEL reagent. Sample slides as negative controls were incubated with the TUNEL reagent in the absence of terminal deoxynucleotidyl transferase. At least 10 fields were selected randomly per animal and the number of apoptotic cells was counted in each field. The results were expressed as an average number of apoptotic cells in ten fields.A terminal transferase dUTP nick end labeling (TUNEL) assay was used for et al.47 using a commercial kit . The activities of MPO in serum samples were expressed as U/L. The cytokines LITAF, IL-1β, and IL-6 in serum were measured by the double-antibody sandwich ELISA method with specific antibodies of LITAF, IL-1β, and IL-6 prepared by Feng et al.20 in our laboratory. Briefly, serum samples were added to each well and rabbit anti-chicken IL-6, LITAF, and IL-1β antibodies were applied as capturing antibodies. The chemical 3,3′,5,5′-Tetramethylbenzidine (TMB) was used as a chromogenic substrate and the color reaction was stopped by sulfuric acid solution. The absorbance values of well plates were read at 450 nm wavelength. The levels of cytokines in serum samples were determined by specific standard curves.The activity of myeloperoxidase (MPO) was determined according to the procedure of Wu P < 0.05 and data are presented as means ± SEM.The data from the experiment were subjected to ANOVA after determination of variance homogeneity by using the SPSS 16.0 software. In this study, gene expression was subjected to the non-parametric Kruskal-Wallis analysis, followed by a Mann-Whitney test and the data are expressed as median and interquartiles. The tests for other parameters were performed using Duncan’s test for multiple comparisons. Differences are considered statistically significant at the level of


Cisplatin, 5-FU and doxorubicin caused similar decreased of cell survival with the increase of let-7d level . All models were resistant to paclitaxel, irrespective of let-7d expression levels. Only two of the generated models (A and C) were radiosensitive (p = 0.0002). Conclusion: the above results indicated that the level of let-7d expression is an important factor for cell response to irradiation and chemotherapeutics.The implication of the let-7 family in cancer development is multifaceted. The family acts as tumor suppressor miRNA although overexpression of let-7 has also been described in many types of cancer, including head and neck squamous cell carcinoma (HNSCC). The aim of this study includes whether different expression levels of let-7d has an influence on chemo- and radiosensitivity. FaDu cell line models with a gradually increased level of let-7d (models from A to E) were generated with the lentiviral system. Expression levels of pluripotency, chemo-radioresistance/apoptosis, and targets of mRNAs were analyzed by real-time reverse transcription-PCR (qRT-PCR). Radiosensitivity was analyzed using a clonogenic assay after irradiation. Response to cisplatin, 5-FU, doxorubicin, and paclitaxel was done with MTT assay. Statistically significant decrease of K-RAS ( Hypopharynx cancer is an uncommon cancer of the head and neck area. It occurs both in males and females, primarily aged between 50 and 60 years. In most cases, hypopharyngeal cancer is of a mucosal squamous cell origin, preceded by various precancerous lesions. Hypopharyngeal cancer does not usually have early symptoms, along with a high ability to metastasize, thus causing the lowest survival rate of all head and neck cancers. The treatment approach is based on surgery and postoperative radiotherapy. These advanced, unresectable cancers are treated by radiation and/or chemotherapy. Personalization of therapy might improve patients’ survival, but it is necessary to find biomarkers describing radio- and chemosensitivity for this kind of tumor. One promising group of biomarkers is miRNAs.In addition, miRNAs are about 22 nucleotides long, with non-coding RNAs being well-known . The miRLethal-7d (let-7d) is a member of the let-7 family, playing a critical role in the regulation of development and carcinogenesis. This family is conserved across species and is considered to be ancient . Let-7 mThis study is based on our observation that in some cases of head and neck squamous cell carcinoma (HNSCC), let-7d is upregulated in tumor tissue . In this2 atmosphere at 37°C. The mycoplasma detection tests (Minerva) were performed routinely during cell line culturing. All cells used in the experiments were the same ones used in the 15th splitting.The FaDu cell line and its variants were maintained in Dulbecco's Modified Eagle Medium (DMEM) with high glucose , supplemented with 8.85% (v/v) of fetal bovine serum (Sigma), 1.77 mM L-glutamine (PAA), 0.885% (v/v) MEM non-essential amino acid solution (PAA), 0.885% (v/v) penicillin-streptomicin (PAA), and 8.85 mM HEPES (Sigma). The 293T cell line used for the production of lentiviral vectors was cultured in DMEM with high glucose (4.5 g/L) medium (Sigma) supplemented with 8.85% (v/v) fetal bovine serum (Sigma) and 20 mg gentamicin (Kirka). Cell lines were cultivated in a humidified 5% CO5’CAATTTAAATGTCATATGGCCAGATA and R1: 5’CATTAATTAAAGTTATCAATGTCAGCA; F2: 5’TCACTCGAGGTCATATGGCCAGATA, and R2: 5’TCACTCGAGAGTTATCAATGTCAGCA (two different pairs of primers to achieve single and double let-7d cassettes). Purified inserts were cloned into pWPXL-GFP plasmids between PacI and SwaI sites. The obtained plasmids were sequenced to verify the correctness of amplified inserts. Lentiviral particles were generated by cotransfecting 20 μg of lentiviral vectors , 15 μg of p89.1 and 7 μg of pMD2G of packing vectors in 293T cells using the CaCl2/HBS method. FaDu-7d, FaDu-7d-7d, and FaDu-GFP cell line models were achieved through lentiviral infection at the single and multiple infection sites (maximum 3 times). The clonal selection was performed.The let-7d gene sequence and its flanking sequences (about 180 nucleotides) were amplified by the PCR method using two pairs of primers: F1: -ΔΔCt method. The snoRNA U18 was used as an internal control.The total RNA from cell line models with different levels of let-7d expression was isolated using the TRI Reagent (Sigma). The miRNA levels were defined by a two-step qRT-PCR method, using TaqMan microRNA Assay . Data obtained were compared to FaDu-GFP cell lines and calculated with the 2The proliferation ratio of cell line models was measured by 3-H thymidine incorporation assay. 7000 cells per well in 96-well dishes were seeded. After 48 h of incubation, the 3-H thymidine was added. Incorporation into DNA was measured with a scintillation counter  and compared to the FaDu-GFP cell line.-ΔCt method. The GAPDH gene was used as a reference, and melting curves discriminated non-specific products of PCR reactions. Each experiment was repeated at least 3 times.The qRT-PCR primers for genes involving pluripotency , chemo-, radio-resistance/apoptosis  and let-7d targets  were designed with the Universal ProbeLibrary Assay Design Center . The reverse transcription PCR was performed using the iScript cDNA synthesis kit  while the qRT-PCR was performed with the 2x SYBR Green master mix  and Light Cycler 96. Results were calculated with the 2Cell line irradiation experiments were performed by the Medical Physics Department. Cells were irradiated on a Varian Clinac 2300 linear accelerator. The cells were irradiated in a special water phantom MP1  with 6 MV accelerating potential.2 culture flasks, which were filled up with PBS, and were irradiated using a dose of 2 Gy. Following that, irradiated and control (non-irradiated) cells were seeded in 6-well plates. The cells were cultivated for 10–14 days. Next, the colonies were stained and counted.The cells were seeded in 25 cm50 of cisplatin, 5-FU, doxorubicin, and paclitaxel for the FaDu cell line was defined using the MTT assay.The ICIC50 concentration of drugs was added and incubated for 48 h with cells. The results for samples treated with the drug were compared to those without drug treatment, as well as being compared to control (FaDu-GFP) line.7000 cells per well of cell line models were seeded in 96-well plates. The p < 0.05 was considered to represent a statistically significant result. The different types of variables  were measured in subsequent A-E cell lines with different levels of let-7d expression; these were analyzed using one-way analysis of variance (the ANOVA test) with either a post-test for trend or a comparison of each cell line to the control. The Bonferroni correction was used for estimation of post-test significance.Statistical analysis was performed with the use of Prism v. 4.0 . All experimental values were obtained in at least three independent experiments and presented on average with standard deviation error bars. A two-tailed Different levels of let-7d expression have been achieved using the lentiviral system. The modified cells were chosen using a clonal selection method, in order to achieve a cell line with a “mother” genetic background. The main limitation of this experimental approach is the unpredictable position of the lentiviral integration: this may affect the expression of neighboring genes. Nevertheless, this system is considered safe and is commonly used for RNAi experiments, as well as for induced pluripotent stem cell (IPS) production. To verify that the side-effects of lentivirus did not affect cell response, we compared cells with single and double cassettes of let-7d. Two FaDu cell lines were created with a single cassette of let-7d (FaDu-7d) and with a double cassette of let-7d (FaDu-7d7d). The second one was generated with the use of half amount of lentivirus with the double let-7d construct for transfection. The FACS analysis of GFP confirmed that FaDu-7d7d contained twice less amount of virus particles. The expression level of let-7d in the both lines was the same, what was reflected with the same behavior after irradiation, . Experimp < 0.0001). Models were ordered from A with the lowest level of let-7d to E as the highest one; in this composition of let-7d expression, a linear trend was shown (p < 0.0001) in p = 0.3739) and a trend in groups was not observed (p = 0.2379). Although model D seemed to behave differently from the others the difference is not statistically significant and therefore the most likely it does not result from biological features.Using the lentiviral system and the single clone selection method, six cell line models  were obtained. The expression level of let-7d for each model was calculated, compared to the FaDu-GFP cell line. The following results were obtained: A: 8.72; B: 17.44; C: 22.85; D:37.93, and E: 57.89. The flow cytometry analysis of the GFP marker showed high homogeneity (over 97% of FITC-H) of positive cells compared to non-transduced FaDu cells . Models p = 0.0369), without a trend and for CASPASE3 . Other let-7d targets mRNA did not show statistically significant differences (p = 0.0111 (but not trend p = 0.344) and ATM p = 0.0506 (but not trend p = 0.203). For ABCB1 expression, differences among models were not statistically significant (p = 0.2029) but a decreasing trend of expression was observed in the group of models (p = 0.034). Expression level of BCL2 did not differ among the analyzed models. This part of the analysis was presented in p = 0.01), ATM (p = 0.044), and positive trends for most analyzed mRNAs: CASPASE3 (p = 0.0017), ATM (p = 0.0090), K-RAS (p = 0.0497), N-RAS (p = 0.0364), HMGA1 (p = 0.0414), and ARID3A (p = 0.0367). It must be noted that although expression of most genes does not show a significant trend (due to high levels between-model variation) when we observe almost all cases, the expression of analyzed genes decreases with increasing levels of let-7d . We expected that increased expression of let-7d (A-E) would show decreased expression of mRNAs. This indicates the expected let-7d’s expression of all studied genes; however, the effect was too small, while the experimental variety was too large, yielding no statistical significance for the individual gene. Data is presented in There were no differences among models for OCT3/4, SOX2 and NANOG mRNAs expression . Analysiferences . In the 50 dose was established using the non-modified FaDu cell line and was as follows: 1.12 μg/mL for cisplatin; 0.86 μg/mL for 5-FU; 0.54 μM for paclitaxel, and 0.06 μM for doxorubicin. Cisplatin, 5-FU and doxorubicin caused a reduction of the proliferation ratio in all models. Cisplatin decreased cell survival by about 20% compared to control in FaDu-GFP (with the exception of model B). The difference in cell survival among models was statistically significant (p = 0.0014) and a linear trend was also observed (p = 0.0460). We noted that 5-FU also caused a decrease of cell survival (except in model B). Differences in cell survival among models were statistically significant (p = 0.004) and the linear trend was present (p = 0.0005). Similar results were obtained for doxorubicin, which generally caused reduction of cell survival (except in model E). Differences among models were statistically significant (p < 0.0001), as we observed a linear trend (p < 0.0001). Models B and E demonstrated a different response to chemotherapeutics. Model B was resistant to cisplatin and 5-FU. Model E was resistant to Doxorubicin. Contrary to the above chemotherapeutics, paclitaxel caused about 40% higher cell survival in comparison to the control in FaDu-GFP, without differences among models (p = 0.1484) or linear trends (p = 0.7651). All models were resistant to paclitaxel irrespective of let-7d expression level. Experimental results are summarized in The cell line models (A-E) were exposed to chemotherapeutics used in HNSCC treatment. The ICp = 0.0002) only for models A and C. The linear trend was not observed. Experimental data is summarized in The cell line models were irradiated by a dose of 2 Gy, culturing 10–14 days, at which point the survival fraction (SF) was calculated. The results for each model were as follows: A—30.71%; B—54.33%; C—36.67%; D—71.36%; E—59.81%, and FaDu-GFP—60.08%. The differences in survival fraction were statistically significant . In the case of patients with esophageal cancer, let-7b and let-7c were used as indicators of sensitivity to cisplatin . Let-7 aIn experiments with irradiation, statistically significant differences in survival fraction were obtained only for models A and C. Generally, let-7 family is overexpressed in radiosensitive cancer cells with the exception of let-7g, which overexpression was associated with radioresistance. It was also postulated that let-7 radiosensitized cells via regulation of the RAS family , 48. ThiThe determination of individual miRNA expression changes can be an important marker of successful patient treatment. We focused on let-7d, known as miRNA suppressor molecule, for which overexpression was described in HNSCC. In other studies both over and underexpression of let-7d in different cancer types were also observed. In fact, the actual level of let-7d differ substantially between cancer samples and cannot be simply classified either as a high (overexpression) or low (underexpression). Therefore in our study, we focused on the question, whether incrementally different levels of let-7d influence chemo- and radiosensitivity. Generally, increased levels of let-7d enhanced cells sensitivity to chemotherapeutics. Only two of the investigated models showed an effect after irradiation. On the other hand, growing level of let-7d expression caused a declining tendency in the expression of the studied genes but it does not let to connect these specific radio- and chemoresistant let-7d levels with changes of expression of investigated mRNAs. Summarizing, we found that level of let-7d was of significance for cell response to irradiation and chemotherapeutics.


Several studies have assessed the clinicopathological and prognostic value of cyclooxygenase-2 (COX-2) expression in patients with head and neck cancer (HNC), but their results remain controversial. To address this issue, a meta-analysis was carried out. A total of 29 studies involving 2430 patients were subjected to final analysis. Our results indicated that COX-2 expression was not statistically associated with advanced tumor stage  but correlated with high risk of lymph node metastasis  and advanced TNM stage . Moreover, COX-2 expression had significant effect on poor OS , RFS  and DFS . The results of subgroup analyses revealed that COX-2 expression was related with high possibility of lymph node metastasis in oral cancer  and advanced TNM stage in oral cancer  and no site-specific HNC . However, subgroup analyses only showed a tendency without statistically significant association between COX-2 expression and survival. Significant heterogeneity was not found when analyzing clinicopathological data, but it appeared when considering survival data. No publication bias was detected in this study. This meta-analysis suggested that COX-2 expression could act as a prognostic factor for patients with HNC. Head and neck cancer (HNC) comprise a series of tumors arising in the lip, oral cavity, pharynx and larynx. It was reported that there were an estimated 387,100 new cases of HNC and 196,200 deaths in 2012 worldwide –6. The pIt is now generally accepted that inflammatory microenvironment plays critical roles in tumor development . The cycPrevious experimental studies have shown that COX-2 has an important role in the growth and metastasis of HNC by a variety of pathways , 19. MorLiterature search and eligibility assessment were performed independently by 2 reviewers and disagreements among them were resolved by consensus. Figure p = 0.074; Figure p = 0.027; Figure p = 0.015; Figure I2 = 0%, p = 0.636 for tumor stage; I2 = 21.6%, p = 0.202 for lymph node metastasis; I2 = 0%, p = 0.505 for TNM stage).The associations between COX-2 expression and advanced tumor stage, high risk of lymph nodal metastasis, and advanced TNM stage were reported by 15, 17, and 14 studies, respectively. Our results showed that COX-2 expression was not significantly correlated with advanced tumor stage ; LC group ; NPC group ; and no site-specific HNC group . These results again indicated that COX-2 expression was not correlated with advanced tumor stage. Subgroup analyses suggested a positive correlation between COX-2 expression and high possibility of lymph node metastasis in patients with OC . But we failed to reveal a statistically significant association in patients with LC , NPC  and no site-specific HNC . For TNM stage, the results of subgroup analyses showed that COX-2 expression was significantly correlated with advanced TNM stage in patients with OC  and no site-specific HNC . However, this correlation was not found in LC group  and NPC group .Subgroup analyses were performed to examine the correlation of COX-2 expression with clinicopathological parameters in different tumor subtypes (Table p = 0.001), indicating that COX-2 expression had a significantly poor survival effect on patients with HNC .As shown in Table C Figure . Similarp = 0.106 for OS); LC group ; NPC group ; and no site-specific HNC group . Despite none of subgroup analyses showed statistically significant association between COX-2 expression and patients' survival, COX-2 expression had a tendency to suggest poor survival of HNC patients as the pooled HRs of all subgroup analyses were greater than 1.We also performed the subgroup analyses to evaluate the prognostic role of COX-2 in patients with site-specific cancers. The combined effect sizes were displayed in Table We assessed the publication bias by visually assessing a funnel plot for asymmetry and by quantitatively performing Begg's test and Egger's test. As shown in Figure The prognostic value of COX-2 expression has been investigated extensively in various cancers –25. Thesp = 0.027) and 1.33 , suggesting that COX-2 expression was associated with tumor progression and metastasis. This is consistent with previous in vitro studies which showed that COX-2 was able to promote the proliferation, migration and invasion of HNC cells [To the best of our knowledge, this is the first comprehensive meta-analysis to confirm the clinicopathological value of COX-2 expression in HNC. The combined OR for COX-2 expression on high possibility of lymph node metastasis and advanced TNM stage were 1.28  for OS, which suggested that patients with COX-2 expression had a shorter overall survival time. In addition, the pooled HR for RFS was statistically significant , indicating that COX-2 expression is a predictor of worse recurrence-free survival. A significant association was also found between COX-2 expression and poor DFS . These results hinted that COX-2 expression could work as a prognostic factor for patients with HNC. Radiotherapy and platinum-based chemotherapy are important treatment options for HNC [p = 0.014, median OS: 249 vs. 152 days; p = 0.02). His studies suggested that inexpensive COX-2 inhibitor might be a good option for palliative chemotherapy in patients with HNC, especially for patients in lesser developed countries. Thus, COX-2 expression correlates with poor survival and targeting COX-2 may be an effective way to control HNC.Twenty studies were enrolled to investigate the effect of COX-2 expression on survival of HNC. We got the combined HR value of 1.93  infection is a well accepted risk factor for the development of OC , 70, whiEstimating heterogeneity and publication bias is an essential part of a meta-analysis. In this study, heterogeneity test revealed no significant heterogeneity when analyzing the associations between COX-2 expression and advanced tumor stage, high risk of lymph node metastasis and advanced TNM stage. But heterogeneity appeared when assessing the prognostic value of COX-2 expression. The heterogeneity may partly come from the variations in assessing COX-2 expression. Although IHC was the only method used to detect COX-2 expression in these studies, large variability was presented when defining COX-2 overexpression. First, some studies defined COX-2 status based on the staining extents, whereas others used a scoring system. Second, the cutoff values for judging COX-2 overexpression varied with studies, ranging from 5% to 50% when the staining extents were used as cut-off points. The standardization of COX-2 overexpression assay may resolve this problem in the future. With regard to publication bias, no clear evidence of funnel plot asymmetry was found by visual assessment. Moreover, no publication bias was detected according to both Begg's test and Egger's test. These findings suggested that our results were robust and not far from the actual situation.However, the present meta-analysis still has several limitations. First, this study is a literature-based meta-analysis, making our results less reliable than individual patient data-based analysis. Second, significant heterogeneity was noted when analyzing the association between COX-2 expression and patients' survival. Third, studies that cannot provide sufficient data to extract OR or HR were excluded. The exclusion of these studies may make our pooled effect sizes differ from their true value on some level.In view of this study, our findings showed that COX-2 expression correlated with high risk of lymph node metastasis and advanced TNM stage in HNC. Moreover, COX-2 expression indicated poor OS, RFS and DFS in patients with HNC. In conclusion, COX-2 expression can act as a prognostic factor for HNC, which might help to define high risk patients and guide clinical decision making. However, there are two important questions that need to be further answered. First, significant heterogeneity was noted when analyzing the impact of COX-2 expression on survival. Second, the results of subgroup analyses were not always consistent with that of pooled analyses. Only a tendency without statistically significant association between COX-2 expression and patients' survival was showed in subgroup analyses. Considering these issues, our results need to be validated and updated in the near future.Electronic searches for relevant articles in PubMed, Embase, and Web of Science databases were conducted in December 2015. The search strategy was generated by combining key words related to COX-2 (‘cyclooxygenase-2′ or ‘COX-2′), HNC , and prognosis (‘prognosis’ or ‘prognostic’). Moreover, we manually searched the reference lists of relevant articles for additional publications.Studies were included in this meta-analysis if they met the following criteria: a) all patients recruited in the study were diagnosed with HNC; b) COX-2 expression was evaluated in primary tumor tissues; c) the clinicopathological or prognostic value of COX-2 expression was tested in the article; d) only English-language studies were included; e) the OR or HR and their corresponding 95% CIs were described or could be statistically extracted from the study; f) When several articles were from the same patient population, the newest or most informative single article was included.The following information was extracted from each study: first author's last name, publication year, tumor types, number of patients, COX-2 expression assay (method and cut-off level), clinicopathological data , and survival data .p < 0.05). OR > 1 or HR > 1 implied a poor survival for the COX-2 expression group. The Cochran Q test and I2 test were performed to assess the heterogeneity between studies. When the Cochran Q test p value was ≤ 0.10 and I2 test I2 value was ≥ 50%, statistically significant heterogeneity was considered to be present. When heterogeneity was absent, fixed effects models were employed; otherwise, random effects models were adopted. Funnel plots, Begg's test, and Egger's test were performed to test publication bias. All analyses were carried out by using Stata Statistical Software, version 12.0 .According to clinical characteristics, T3 and T4 were combined as advanced tumor stage; TNM stage III and stage IV were combined as advanced TNM stage. OR and its 95% CIs were used to describe the correlation between clinicopathological factors and COX-2 status. HR and its 95% CIs were adopted to estimate the prognostic value of COX-2 expression. The individual OR or HR estimates were combined into an overall OR or HR and the results were presented graphically in the form of a forest plot. Pooled effect sizes were considered to be significantly different if their 95% CIs did not include 1 (


Computational analysis of protein-protein interaction provided the crucial information to increase the binding affinity without a change in basic conformation. Several docking programs were used to predict the near-native poses of the protein-protein complex in 10 top-rankings. The universal criteria for discriminating the near-native pose are not available since there are several classes of recognition protein. Currently, the explicit criteria for identifying the near-native pose of ankyrin-protein complexes (APKs) have not been reported yet.In this study, we established an ensemble computational model for discriminating the near-native docking pose of APKs named “AnkPlex”. A dataset of APKs was generated from seven X-ray APKs, which consisted of 3 internal domains, using the reliable docking tool ZDOCK. The dataset was composed of 669 and 44,334 near-native and non-near-native poses, respectively, and it was used to generate eleven informative features. Subsequently, a re-scoring rank was generated by AnkPlex using a combination of a decision tree algorithm and logistic regression. AnkPlex achieved superior efficiency with ≥1 near-native complexes in the 10 top-rankings for nine X-ray complexes compared to ZDOCK, which only obtained six X-ray complexes. In addition, feature analysis demonstrated that the van der Waals feature was the dominant near-native pose out of the potential ankyrin-protein docking poses.http://ankplex.ams.cmu.ac.th.The AnkPlex model achieved a success at predicting near-native docking poses and led to the discovery of informative characteristics that could further improve our understanding of the ankyrin-protein complex. Our computational study could be useful for predicting the near-native poses of binding proteins and desired targets, especially for ankyrin-protein complexes. The AnkPlex web server is freely accessible at The online version of this article (doi:10.1186/s12859-017-1628-6) contains supplementary material, which is available to authorized users. Due in vitro  and in somplexes , 10. TheProtein–protein docking is a well-known method for generating protein–protein complexes (poses) using computational methods. The challenging task of identifying the exact bound state of a pair of proteins must consider the following factors: (i) there are several potential ways that a pair of proteins can interact, (ii) the flexibility of the protein, and (iii) changes in the protein conformation after binding . Currentvia assessing the CAPRI criteria in the 10 top-rankings based on the features of ZDock, ZRank, or E_RDock. However, successful predictions do not occur for all cases [ZDOCK has successfully predicted several near-native complexes (poses) of antibody-antigen, enzyme-inhibitor and other pairings ll cases –20. Morell cases . TherefoThe near-native docking pose of Ankyrin-Her2 was successfully predicted using ZDOCK and an extra scoring function . RecentlIn this study, we made a systematic attempt to develop a computational approach for achieving near-native predictors in 10 top-rankings of ankyrin-protein docking poses, which we named AnkPlex. Moreover, this method was generated for (i) analysing and characterizing ankyrin-protein complexes by using a set of informative features that have potential applications and (ii) establishing a user-friendly web server to obtain the desired results without the need to follow complicated mathematical equations generated by the research scientist. The docking poses of seven X-ray complexes of APKs, which had ankyrins with 3 internal domains, were generated using the reliable docking tool ZDOCK. The construction of the docking poses calculated by PSC alone and summation of PSC + DE + ELEC demonstrated there were different numbers of near-native docking poses. The steps for AnkPlex establishment included (i) balancing the near-native and non-near-native poses; (ii) processing the dataset through machine learning of a decision tree algorithm (DT) and a logistic regression (LG) with a combination of 11 features; (iii) selecting the efficient predictive models of DT and LG; and (iv) processing the dataset by combining models of DT and LG.Protein Data Bank (PDB) database for 41 APKs reported up to May 2014. Analyses of the 41 APKs were performed through data pre-processing using the following steps: (i) APKs containing 3-internal-domain were included; (ii) redundant APKs were excluded; (iii) APKs were filter based on the recognition areas [1), complex 2 (C2), complex 3 (C3), complex 4 (C4), complex 5 (C5), complex 6 (C6), and complex 7 (C7). At the same time, the rest of the APKs, including unknown 1 (U1) and unknown 2 (U2), were designated the test group (Ank-TEST). In order to avoid the distinct results from the different selections of training and test sets, other 35 possible datasets were constructed and were used to generate the predictive models for the identification of near-native poses.X-ray crystal structures of ankyrin-protein complexes (APKs) were collected from the on areas ; and  is the Pairwise Shape Complementarity (PSC) score and it was optionally augmented with the electrostatics (ELEC) and the desolvation energy (DE). In this study, the α and β have the default values of 0.01 and 0.06, respectively.ZRankElec (B) is the long-range electrostatic energy and the only fully charged side-chain, as represented in the following equation:qi and qj are the charges on ankyrin and the protein atoms, respectively. The rij in the equation stands for the distance between the atoms of ankyrin and the protein.ZRankSolv (C) is the desolvation term based on the Atomic Contact Energy (ACE).aij is the ACE score.ZRank (D) is a linear combination of ZRankVdw, ZRankElec, and ZRankElec.ZRankVdw score was calculated as follows:ZRankVdw (E) is the van der Waals and short-range electrostatics energy with a distance between the atom pair being less than 5.0 Å. This calculation was based on the parameters of the CHARMm 19 polar hydrogen potential. The εij and σij are the depth and the width, respectively, of the coefficient for the CHARMm 19 polar H.st and the 2nd minimized structure of the ankyrin-protein docking poses, respectively.E_vdw1 (F) and E_vdw2 (H) are the van der Waals energy, as presented in Equation (5), of the 1st and the 2nd minimization, respectively.E_elec1 (G) and E_elec2 (I) are the electrostatic energy, as presented in Equation (2), of the ankyrin-protein docking poses processed for the 1nd minimization of the ankyrin-protein docking poses.E_sol (J) is the desolvation energy, as shown in Equation (4), of the 2E_RDock (K) is the summation of E_sol and (0.9 × E_elec2).Based on observations of the generation of the features, ankyrin-protein docking poses were generated for the energy features using the ZDOCK protocol , 14 (a s1-C7). As shown in Additional file A1: Table S5, SVM yielded 100% near-native poses in the internal testing sets but could not obtain any near-native pose in the external testing sets. The DT and ANN provided the near-native poses from both internal and external testing sets. According to a dataset of C5, the DT was superior in achieving the near-native poses of internal testing sets than the ANN. The LG provided a weighted summation that could rank the docking poses to achieve the near-native poses in the 10 top-rankings. As a consequence, the DT and the LG were selected to construct an ensemble model.Several learning models were constructed including decision tree (DT), logistic regression (LG), artificial neural network (ANN), and support vector machine (SVM) using Ank-TRN  and a logistic method (LG). The decision trees and the logistic regression methods were selected due to the fact that they provide a high number of predicted positive values (true near-native poses). The logistic regression especially provided a weighted summation that was finally ranked to search for near-native poses in 10 top-rankings. All 11 features and all datasets were used to build the DT and LG models. The Ank-TRN (7 APKs) and the Ank-TEST 2 APKs) were evaluated by AnkPlex using the following steps, as shown Fig.  APKs wer1. The number of near-native poses and non-near-native poses were balanced. ZDOCK using PSC + DE + ELEC and regKp provided 699 near-native poses and 44,334 non-near-native poses of Ank-TRN. The non-near-native poses were randomly clustered into 65 groups . Therefore, each training set was composed of the same near-native poses and different groups of non-near-native poses.2. A predictive model using the DT and the LG models was established. All 11 features were combined and generated as feature subsets . The total number of feature subsets was calculated to be 4,095 by following this equation:The DT model was established from 4,095 feature subsets and 65 training sets using the J48 algorithm , 27. The3. An efficient predictive model of the DT and the LG models was selected. Ank-TRN consisted of 7 APKs and was submitted to the learning method for predicting the near-native poses. True positive rates (TPrate) greater than 50% were used as the cut-off value for an efficient learning method. The learning methods that demonstrated a TPrate greater than 50% were selected to further establish an ensemble learning model.DT) and the LG-based learning models (OLMLG) from Step 3 for reducing the number of non-near-native docking poses. The main process of the proposed method, AnkPlex, for increasing the number of TPs (reducing non-near-native poses) consisted of the following steps: (i) only predicted positive samples (PPVDT) derived from OLMDT were select, (ii) a logistic score (LGS) on PPVDT using the LG model was calculated, and (iii) PPVDT was ranked according to LGS and the 10 top-ranking poses that demonstrated the highest LGS were selected. The near-native pose(s) or the true positive (TP) in the 10 top-ranking poses were our targets. The summation score of AnkPlex was defined in the equation given as Equation (7) on Ci, where i = 1, 2, …, 7, and Yi would be set as 1 in case TP was found in the 10 top-ranking poses. Otherwise, Yi would be set as 0. Finally, the score of AnkPlex was the summation product, as defined in the following equation:PP belongs to Ank-TRN containing seven complexes  and Yi would be set as 1 when TP appears in the 10 top-ranking poses. Otherwise, Yi would be set as 0. The number of #PPTRN indicated the sample of Ank-TRN in which the LGS score was among the 10 top-ranking near-native poses. The number of #PPTEST showed the LGS score of the Ank-TEST was among the 10 top-ranking poses.4. Ensemble methods were established. The ensemble learning method, named AnkPlex, was constructed by randomly integrating the DT-based learning models . The method validation parameters, including accuracy (ACC), sensitivity (SEN), and precision (PRES), were calculated using the following equations:There were a few ankyrin-protein complexes (APKs) reported in the PDB database. Forty-one ankyrin complexes, with the number of internal domains ranging from 2–7, have been reported . The highest number, 19 complexes, of ankyrin-proteins contained 3 internal domains. Furthermore, the APKs that reacted with the target using recognition areas were selected. Focusing on target proteins, only proteins with common folding structures, i.e., alpha-, beta- and alpha-beta structures, were considered. Therefore, nine complexes, which included 1SVX, 4ATZ, 3Q9N, 1AWC, 2BKK, 2Y1L, 4DRX, 2P2C, and 4HNA, were used in this study. These nine complexes were randomly divided into two groups, i.e., 7 complexes as Ank-TRN and 2 complexes as Ank-TEST.To optimize the ZDOCK calculation, X-ray crystal structures of APK-TRN, including seven APKs, were calculated with different feature calculations, including the PSC and the PSC + DE + ELEC. The total number of docking poses, including near-native and non-near-native poses, was 54,000 poses (54Kp). Subsequently, the numbers of near-native poses calculated by PSC and PSC + DE + ELEC were compared. As shown in Table p-value <0.001. As presented in Table p = 0.178) and E_vdw2 (p = 0.010). Subsequently, 11 features calculated with the ZDOCK calculation were applied to establish the learning methods.According to the ZDOCK calculations of Ank-TRN, 11 features of near-native poses and non-near-native poses were generated. Univariate statistical approaches were employed to perform exploratory data analysis using average and standard deviations for summarizing important patterns. As shown in Table DT) and the LG-based learning models (OLMLG) were established using the 4,095 feature sets. The learning methods demonstrated the average of the true positive rate to be greater than 50% (TPrate ≥ 50%), and consisted of 4,762 OLMDT and 2,688 OLMLG. The learning models that represented TPrate ≥ 50% with the 10 top-ranking poses of %ACC are shown in Table DT) and Table LG). As a result, ABDEHIJK_g14 of OLMDT exhibited the highest %ACC with %TPrate ≥ 50%. This learning method consisted of sequential combination feature sets that included ZDock (A), ZRankElec (B), ZRank (D), ZRankVdw (E), E_vdw2 (H), E_elec2 (I), E_sol (J), and E_RDock (K) calculated from non-near-native dataset number 13. In addition, CDFGJ_g10 of OLMLG also demonstrated the highest %ACC with %TPrate ≥ 50%. The percentage of precision (%PRES) for all the 10 top-ranking poses of OLMDT and OLMLG was low, which indicated that there was a high number of false positive results (FP). To diminish the number of FP, only the 10 top-ranking poses based on the ZRank score were selected to represent the true positive poses (TP). If the TP were found in 10 top-ranking poses from each Ank-TRN, #PP was designated 1. Thus, the #PP-values of seven Ank-TRN (#PPTRN) were in the range of 0 to 7. As shown in Tables TRN of OLMDT and OLMLG were only 6. Therefore, the individual learning method of OLMDT or OLMLG was not capable of providing the maximum value for #PPTRN.Eleven features of each of the near-native poses (669) and non-near-native poses 44,334) calculated from Ank-TRN based on the recognition areas (regKp) were used to establish the learning methods. Based on the unbalanced number of docking poses, training sets were generated by clustering the non-near-native poses and the near-native poses into 65 sets . Eleven features were calculated from each training set and were ordered to generate 4,095 feature sets. The DT-based learning models  and 2,688 of the LG-based learning models (OLMLG) were randomly combined to generate an ensemble model. Interestingly, the combination of the ensemble model from ABEHIJ_g56 of OLMDT and CDFGHJ_g30 of OLMLG demonstrated superior prediction efficiency due to the fact that this ensemble model (ABEHIJ_g56- CDFGHJ_g30) achieved maximum values for #PPTRN and #PPTEST of 7 and 2, respectively. Therefore, the ensemble model, ABEHIJ_g56- CDFGHJ_g30, was designated to be an ensemble computational model for predicting the near-native docking pose of APKs or “AnkPlex”  and OLMLG (CDFGHJ_g30) provided a #PPTRN value of 6. The first TP of C5 predicted by ABEHIJ_g56 and the C6 predicted by CDFGHJ_g30 were found at pose numbers 14 and 19. This result indicated that a single learning model could not produce all the true positive poses. In the case of the Ank-TEST, OLMDT could not provide the value for the #PPTEST, whereas the #PPTEST of OLMLG was comparable to AnkPlex. Consequently, it can be concluded that the ensemble model, AnkPlex, was capable of including a #PPTRN value of 7 and a #PPTEST value of 2, which suggested that the prediction efficacy of AnkPlex was superior to the single learning model. In addition, the predictive models generated from other 35 possible datasets demonstrated the average number of #PPTRN and #PPTEST value of 6.78 ± 0.42 and 2 ± 0.00, respectively. This indicated that different selections of training and test sets had no effect in the generation of the learning models for predicting the near-native poses.To enhance the prediction efficacy of the generated learning methods, 4,762 of the DT-based learning models  of HIV-1 [GAG1D4 was already constructed. However, the complex structure of AnkGAG1D4-CANTD was not detected [GAG1D4-CANTD and performed re-scoring with AnkPlex. The results revealed that three near-native structures of AnkGAG1D4-CANTD were found in the 10 top-rankings. The recognition residues of AnkGAG1D4-CANTD interactions were further investigated by observing interacting distances ≤ 5 Å. As a result, one docking pose showed that residue R18 was located on the recognition areas of CANTD and two docking poses demonstrated residues R132 and R143 played key roles in the interaction with AnkGAG1D4 (data not shown). This result correlated with previous ELISA results. A point mutation of R18A on helix 1 and R132A and R143A on helix 7 of CANTD showed negative binding to AnkGAG1D4. Thus, R18, R132 and R143 were the key residues of CANTD binding to AnkGAG1D4 [GAG1D4 using this learning model, AnkPlex could not only discriminate the near native docking poses of AnkGAG1D4-CANTD complex but also demonstrated the correct orientation of the recognition area to CANTD.To apply the AnkPlex for investigating the ankyrin-protein complex, AnkR values) were used to identify the correlation between LGS and the weights of the six features to obtain the near-native poses. As shown in Fig. R values of the six features consisted of ZRank (R = 0.60), ZRankSolv (R = −0.56), and E_sol (R = 0.54), which indicated that these three features played an important role in the AnkPlex model for distinguishing near-native poses.Identification of informative features among the 11 features was critical for designing a powerful learning model and for understanding and obtaining insights into the ankyrin-protein docking poses. Based on the six features (CDFGHJ) used in the calculation of the LGS in AnkPlex, the Pearson correlation coefficients . The characteristics of the ZRank score between the near-native and the non-near-native poses were significantly different, with p < 0.001, as shown in Table DT(ABEHIJ_g56)–OLMLG(DFGHJ_g30) was able to obtain #PPTRN = 1 and #PPTEST = 0. However, AnkPlex (OLMDT(ABEHIJ_g56)–OLMLG(DFGHJ_g30) achieved success with #PPTRN = 7 and #PPTEST = 2. Therefore, ZRank was concluded to be an important feature of AnkPlex due to the fact that it could enhance the predictive performance of near-native poses.The ZRank score was ranked as the 1Since ZRank is a linear combination of van der Waals (ZRankVDW), electrostatics (ZRankElec), and desolvation energy (ZRankSolv), one of them had to be identified as the most important. From Additional file nd and the 3rd informative features with R values of −0.56 and 0.54, respectively. Moreover, these two features showed differences in their characteristics between near-native and non-near-native poses, with p < 0.001, as shown in Table DT(ABEHIJ_g56)–OLMLG(CFGHJ_g30) provided #PPTRN = 6 and #PPTEST = 1. Similar to E_sol, the performance of AnkPlex lacked E_sol, i.e., (OLMDT(ABEHIJ_g56)–OLMLG(CDFGH_g30)) yielded #PPTRN = 6 and #PPTEST = 1. This result indicated that the absence of ZRankSolv and E_sol slightly reduced the predictive performance of AnkPlex. However, these two features were required for predicting near-native poses. ZRankSolv was a component of ZRank. This outcome emphasized that desolvation was important for obtaining near-native poses in the ankyrin-protein interaction. Additionally, it was also required for the accuracy of other protein–protein complexes [ZRankSolv and E_sol were the desolvation energies estimated by the summation of the Atomic Contact Energy (ACE) in which the difference between the two features was in the force field calculation and side chain orientation. ZRankSolv and E_sol were the 2LGCFGHJ_g providedomplexes .The LGS score was a combination of the energy determined in the interaction area between ankyrin and proteins of ≤5 Å. In AnkPlex, the interaction area on ankyrin was located on variable and conserved residues of the L-shaped repeat belonging to the internal repeats, the N-terminal repeat and the C-terminal repeat . The funAccording to the hydrophobicity on the interface in AnkPlex  is freely available online.An ensemble method, named AnkPlex, was constructed for fast prediction of near-native states of ankyrin-protein complexes. The AnkPlex model was constructed based on a combination of features generated from the ZDOCK program without using manual inspections. AnkPlex successfully obtained the near-native poses of nine ankyrin-protein complexes in the 10 top-ranking poses. ZRank, which is a combination of electrostatic, desolvation, and van der Waals energy, was the most important feature in AnkPlex. In addition, van der Waals was the dominant feature for obtaining the near-native docking poses. To develop the method for predicting near-native poses of protein complexes, we have implemented easy access to the best models for the scientific community on a web server. AnkPlex  of the nine ankyrin-protein complexes. Table S5. The percentage of predictable true near-native poses of the internal and external testing sets on leaning methods of decision tree (DT), logistic regression (LG), artificial neural network (ANN), and support vector machine (SVM). (DOC 125 kb)Informative characteristics of nine ankyrin-protein complexes. Additional file 2:Supplementary datasets used in this study. (XLS 13646 kb)


We then delineate the outcome of the co-cultures when equal distributions of resources were provided. The growth behavior of the co-culture was found to be dependent on the types of microbial species present, their specific metabolic activities, and resulting changes in the culture environment. Through this reductionist approach and using novel in vitro combinations of microbial species under anaerobic conditions, the results of this work will aid in the understanding and design of synthetic community formulations.Network analysis of large metagenomic datasets generated by current sequencing technologies can reveal significant co-occurrence patterns between microbial species of a biological community. These patterns can be analyzed in terms of pairwise combinations between all species comprising a community. Here, we construct a co-occurrence network for abundant microbial species encompassing the three dominant phyla found in human gut. This was followed by an  The nature of microbe-microbe interaction ranges from antagonism to mutualism, and determines the overall composition and function of a microbial community . For insin vitro conditions.Advancement in sequencing technologies has generated an abundance of data on the composition of microbes that live in and on human body . Typicalin-silico predictions from the resulting co-occurrence network, we co-cultured i) Bifidobacterium adolescentis from the Actinobacteria phylum, together with Bacteroides thetaiotaomicron from Bacteroidetes, both of which are known polysaccharide degraders; ii) Faecalibacterium prausnitzii together with Roseburia inulinivorans, both from the Firmicutes phylum and well-known butyrate producers in the human gut [in vitro growth medium. These four species have gained interest as several human gut metagenomics studies have shown that their abundance is associated with healthy gut microbiome, maybe because their genetic flexibility allows them to survive in various ecological niches. Therefore, data on these species would contribute to our understanding of human gut microbiome as they also belong to the core microbiome [Bacteroides thetaiotaomicron is of great importance in terms of symbiotic bacteria-host relationships within the human intestine, as well as for its potential to break down plant polysaccharides [Bifidobacterium adolescentis is a key microbe of the adult-associated Bifidobacteria [In order to determine statistically significant co-occurrence patterns, sufficiently large sample sizes are needed. We therefore used a gut microbial gene catalogue, which was constructed from four different studies conducted over three continents ,7,12–14.uman gut . We choscrobiome . In partcharides . It alsocharides . Bifidobbacteria . Apart fbacteria .http://www.metabolicatlas.com/medusa). Assuming the limitations of available correlation analysis methods, we chose two suitable methods with respect to our dataset. To generate co-occurrence networks, we employed and compared two known methods: i) Spearman correlation [782 human gut shotgun metagenomes from four different studies, Sweden , MetaHITrelation , which hrelation  which harelation . Spearmarelation . The thirelation . LGL appBifidobacterium adolescentis L2-32, Faecalibacterium prausnitzii A2-165 and Roseburia inulinivorans A2-194 (DSM 16841) were kindly provided by Dr. Karen Scott . Bacteroides thetaiotaomicron 29148 was ordered from ATCC. All bacterial strains were maintained at 37°C in Hungate tubes  under oxygen-free CO2 in yeast extract, casitone and fatty acid (YCFA) medium [) medium  and cult3 (Merck), 0.045 g K2HPO4 (Merck), 0.045 g KH2PO4 (Merck), 0.09 g NaCl (Merck), 0.09 g (NH4)2SO4 (Merck), 0.009 g MgSO4 · 7H2O (Merck), 0.009 g CaCl2 (Merck), 0.1 mg resazurin (Sigma-Aldrich), and 1 mg hemin (Sigma-Aldrich). In addition, the final concentrations of following short-chain fatty acids (SCFA) were included : acetate (33 mM); propionate (9 mM); isobutyrate, isovalerate, and valerate (1 mM each). Finally, the volume was adjusted with dH2O in a conical Erlenmeyer flask. The medium was boiled in a microwave to dissolve the complex compounds. After the medium cools down, it was supplemented with cysteine (0.1 g), followed by boiling under oxygen-free CO2 atmosphere for 3–4 minutes. After autoclaving at 120°C for 15 min, filter sterilized solutions of vitamins  per 100 ml of medium. All SCFAs and vitamins were purchased from Sigma-Aldrich. The final pH of medium was maintained to 7.2 ± 0.1.The composition of the base medium per 100 ml, is as follows: 0.25 g yeast extract (BD), 1.0 g casitone (BD), 0.4 g NaHCOB. adolescentis L2-32 and B. thetaiotaomicron ATCC 29148 co-culture experiment. Short chain fatty acid (SCFA) mix was added to YCFA medium, but not to YCGMS medium, as they do not consume them. All other components were the same between the YCFA and YCGMS media.In YCGMS medium, carbon sources such as glucose (G), maltose (M), cellobiose (C), and soluble starch (S) were added to a final concentration of 0.2% (wt/vol) of each. We selected starch as a substrate because it serves as the major source of carbon and energy for the gut microbiota ,27,28. TF. prausnitzii and R. inulinivorans, the bacteria were inoculated into a medium that contained yeast extract, casitone, glucose and disaccharide  with the addition of SCFA mix (YCFAGD medium) and without the addition of SCFA mix (YCGD medium). It has been found that acetate produced from other bacteria in a community enhance butyrate formation by Faecalibacterium prausnitzii through cross-feeding [For experiments with -feeding . Hence, 2 in the gas phase. Each bottle was inoculated with 2% (vol/vol) inoculum. The bacteria were allowed to grow by inoculating equal volumes of inoculum in both the mono- and co-culture respectively. Media without supplementation of inoculum were used as control. The bottles were incubated at 37°C for a maximum of 56 hours, and each experiment was performed in triplicates.Each individual bacterial species was sub-cultured on an agar plate with YCFA medium  in an anGenomic DNA was isolated from a pellet, which was obtained after centrifuging 1 ml of microbial culture at 16 000 rpm for 5 min, using the NucleoSpin® Soil kit . The manufacturer’s instructions were followed, with the exception that the vortex step was replaced with repeated bead beating twice at 5.0 m/s for 60 seconds using the FastPrep®-24 Instrument , with 5 minutes of incubation on ice after each round of bead-beating.B. adolescentis, R. inulinivorans and F. prausnitzii [B. thetaiotaomicron [8 to 100 copies. Later, the obtained 16S rRNA values were normalized with respect to each species’ 16S rRNA copy number.To quantify the abundance of the two-bacterial species in co-culture, we employed 16S rRNA quantitative PCR using a CFX96 Real-Time System (Bio-Rad Laboratories). Samples were analyzed in a 25-μl reaction mix consisting of 12.5 μl 1xSYBR Green Master Mix buffer , 0.2 μM of each primer, and 5 μl of template DNA, with water as a control. qPCR was performed as reported previously: usnitzii  and for aomicron . Standar2SO4 at a flow rate of 0.6 ml/min at 65°C. To quantify the consumption of starch, 1 ml of bacterial cultures grown in YCGMS media were filtrated and a mild post-hydrolysis method were applied. H2SO4 (4% volume of solute/volume of solution) was used to depolymerize starch into its monomer constituents. The samples were incubated at 120°C for 30 minutes. Quantification of the metabolites was performed as previously reported [One-millilitre of homogenized microbial cultures were concentrated by centrifugation  and the supernatant was used for analysis. Glucose, disaccharides, acetate, butyrate, lactate, formate, succinate and propionate were measured by high pressure liquid chromatography (HPLC). For this analysis, the RI and UV detectors and an Aminex HPX-87H column  were used to separate and quantify all the metabolites in the supernatants of the cultures. The column was eluted with 5 mM Hreported .B. thetaiotaomicron was grown for 24 h in YCFA medium as described above. Around 12 h, the pH of the medium was lowered to pH 3.5 with 6 M HCl. Six hours later; the pH of the medium was increased to pH 8.5 with 5 M NaOH. In parallel to this, a similar set of experiments was performed in which the pH range and the order of pH adjustment were altered. Around 12 h, the pH of the medium was increased to pH 6.5 with 5 M NaOH. Six hours later, the pH of the medium was lowered to pH 5.5 with 6 M HCl. Culture media without pH change, and media without inoculum were used as controls. Equal volumes of inoculum were inoculated into each of the samples, except the cell-free control. Each set of experiments was performed in duplicates.Results plotted are the mean values with error bars corresponding to the standard deviation of the biological replicates at each time-point. Statistical comparison of 16S rRNA abundance data between mono-culture and co-culture of each species in same or different medium was evaluated by Student’s t-test using GraphPad Prism version 7.00 for Mac, GraphPad Software, La Jolla California USA. Significance was set at p-value of less than 0.01 (two-tailed).Bacteroides pectinophilus ATCC 43243 was the only exception, as its abundance negatively correlated with other Bacteroides species, and positively correlated with Bifidobacteria , this was the case, we grew Bifidobacterium adolescentis and Bacteroides thetaiotaomicron in mono- and co-cultures in vitro. The pH profile and extracellular metabolite concentrations were measured to evaluate their fermentation activity. Our data showed that B. adolescentis exhibited no significant difference in abundance  and fermentation activity in both the mono- and co-culture conditions . Growth of B. thetaiotaomicron reached saturation around 4 h and 8 h in the co-culture and mono-culture, respectively , whereas R. inulinivorans exhibited significant differences in 16S rRNA copies between the mono-cultures and co-cultures of different medium . Glucose and disaccharides were consumed at similar rates between the mono- and co-cultures in the medium with added acetate  apparently created a niche that could be exploited by low-pH tolerant B. adolescentis. It is well known that many species from the genus Bacteroides are less acid-tolerant, however, there are a few which can grow at low pH [Bacteroides thetaiotaomicron and Bifidobacterium adolescentis could not continue to grow together at their optimal level due to SCFA-driven changes in pH of the environment. In addition, we note from previous studies, that combinations of microbial genera at the species or strain level between the gut commensals could possibly be considered as a factor contributing to the variation and coexistence of species in a community.Previous studies have investigated the growth of several different strains of genera Bifidobacterium and Bacteroides co-cultured in the presence of different single carbon sources , and generally showed higher population levels of acterium . Based ot low pH ,38, henct low pH . AlthougF. prausnitzii and R. inulinivorans can utilize acetate as a co-substrate from other acetate producing gut bacteria [in vivo, it is expected that acetate would be found at high concentration in the gut. We therefore explored two media conditions in order to investigate the co-occurrence pattern of these two butyrate-producers in a hypothetical focal area where there is not sufficient acetate. Presence and absence of acetate in the media demonstrated how changes in SCFA metabolism affect the production levels of different SCFAs. Specifically, how F. prausnitzii shifts between alternative pathways and produces varying proportions of fermentation products  depending on the environmental conditions present. In the acetate-free medium, there was decreased butyrate production by F. prausnitzii, whereas acetate abundance exhibited a smaller effect on the metabolism of R. inulinivorans. This difference in marginal growth corresponds to a minimal dependence upon acetate for growth and metabolism by F. prausnitzii [R. inulinivorans appears to produce enough acetate on its own to initiate further conversion of this metabolite to butyrate. In conclusion, we show that both species can coexist with marginal difference from their respective mono-cultures despite different levels of SCFA production and pH variation in YCFAGD and YCGD media.In a densely populated environment such as the gut, there will be many species that prefer similar conditions and substrate preferences. Nonetheless, this does not necessarily equate to positive correlation, as they would be competing with each other to obtain and meet their similar requirements. This hypothesis led us to investigate the co-occurrence patterns between two species that share similar metabolic pathways and which have not been studied previously in the context of gut bacteria. It has been shown that butyrate producers like bacteria ,39–41. Tbacteria . Given tusnitzii . In contin-silico predictions from metagenomic data sets with experimentally verified microbial patterns would be invaluable to progress in the area of microbial relationships. Overall, we demonstrated how co-occurrence networks can be used to identify species pairs of interest, and experimentally showed their growth and metabolic dynamics in mono- and co-culture conditions. From the co-occurrence network analysis of abundant bacterial species, it was of great interest to investigate and verify the positive co-occurrence pattern between species  of similar metabolic type as predicted from the network, as they would be expected to compete to meet their similar metabolic requirements. While Bifidobacterium and Bacteroides were predicted to co-occur in a negative fashion even when their metabolic pathways of producing few of the major short chain fatty acids were different from each other. These findings are of value in the design of synthetic microbial communities. Top-down approaches such as this setup will facilitate the selection of bacteria when formulating a community, in order to enable a stable co-existence of multiple bacterial species. Likewise, designing synthetic microbial community with a finite number of species to test the difference in bacterial correlations between the healthy and diseased groups in mouse models may reveal the link between microbial factors and disease susceptibility.Provided the significance of correlation networks, complementation of the S1 Fig10 16S rRNA gene abundance and pH of B. thetaiotaomicron during growth with a pH decrease performed at 12.5 h of fermentation, followed by a pH increase at 18.5 h. B. Log10 16S rRNA gene abundance and pH of B. thetaiotaomicron during growth with a pH increase initiated at 12.5 h of fermentation, followed by a decrease performed at 18.5 h of fermentation. Experiments were performed in duplicates. The values represent the mean of each biological replicate.A. Log(TIFF)Click here for additional data file.S2 FigF. prausnitzii and R. inulinivorans mono- and co-cultures in YCFAGD and YCGD medium for 50 h. FR = co-culture of Faecalibacterium prausnitzii and Roseburia inulinivorans, Fpr = mono-culture of F. prausnitzii, Rin = mono-culture of R. inulinivorans. Experiments were performed in triplicates and error bars represent the standard deviation between each biological replicate.A-B. Glucose and C-D. Disaccharide consumption, E-F. Lactate and G-H. Formate production and I-J. pH profile of (TIFF)Click here for additional data file.S3 Fig’ respectively.Experiments were performed in triplicates and error bars represent the standard deviation between each biological replicate. P-values less than and greater than 0.01 are summarized with two asterisks and ‘non-significant (ns)(TIFF)Click here for additional data file.


Atrial fibrillation (AF) is an irregular and often rapid heart rate that can increase the risk of stroke, heart failure, and other heart-related complications. Its incidence increases with age and the presence of concomitant heart disease. We present the cases of a 93-year-old woman, an 82-year-old man, and an 87-year-old woman who developed AF tachycardia. This report highlights the use of a bisoprolol transdermal patch to treat AF tachycardia in 3 adult elderly patients. In this paper, we report an initial treatment strategy using a bisoprolol transdermal patch and show heart rate trends for 24 hours. AF sve drugs . Howeverve drugs . Moreoveve drugs . BisoproThis 93-year-old woman, a resident of a special elderly care nursing home, was referred to our hospital for the treatment of cellulitis. She related a history of treatment for hypertension and atrial fibrillation (AF). Upon arrival, her blood pressure (BP) was 119/83 mmHg and heart rate (HR) was 82 bpm. An electrocardiogram (ECG) demonstrated AF and a complete right bundle branch block . During An 82-year-old man was admitted to our hospital for the treatment of ileus. He related a history of treatment for persistent AF and hypertension. He was administered bisoprolol fumarate tablets (2.5 mg/day) for AF before admission. His HR on admission was 87 bpm. However, he could not take oral medicine because of fasting for treatment of ileus. After 3 days of fasting, he developed AF tachycardia and his HR increased to 150 bpm. Bisoprolol transdermal patch (4 mg) was applied to his chest. This dose was equal to a 2.5 mg bisoprolol fumarate tablet. His HR trends were significantly decreased after 8 hours of bisoprolol transdermal patch, and the frequency and duration of AF decreased until 24 hours after administration .An 87-year-old woman was referred to our hospital for treatment of AF with palpitation and dyspnea . She relβ-blockers, nondihydropyridine calcium channel blockers, digoxin, and certain antiarrhythmic drugs, including amiodarone and sotalol, have been evaluated for efficacy in attaining rate control [β-blockers are the most commonly used drugs to control the ventricular rate during AF [β-blockers also have an antiarrhythmic effect due to the suppression of sympathetic activity [AF is an irregular and often rapid heart rate that can increase the risk of stroke, heart failure, and other heart-related complications. Its incidence increases with age and the presence of concomitant heart disease . Rate co control . β-blockuring AF . β-blockactivity . By reduBisoprolol, also available as a transdermal patch in Japan, is indicated for the management of hypertension . HoweverThe current cases may provide physicians with an initial treatment strategy for the use of a bisoprolol transdermal patch for AF tachycardia patients and especially in elderly patients who are relatively difficult to treat with oral medication.